Stabilization treatment of collagen scaffolds

Abstract

To disclose shape-stabilized collagen scaffolds and methods of obtaining such stabilized scaffolds.SOLUTION: Stroma can be harvested, for example, from human or porcine corneal stroma. The stroma can be shaped during excision or in a separate step after excision. Following shaping (and preferably decellularization), the excised stroma portion is subject to pressure, force or vacuum to reduce fluid content and then irradiated or otherwise treated to induce crosslinking of collagen chains or fibrils 16. Various sources of energy can be employed to induce peptide bond crosslinking of collagen including, for example, ultraviolet (UV) radiation. The scaffolds can also be selectively densified or patterned. The invention is particularly useful in forming stable lenticules 10A for intracorneal implantation in additive ocular surgery.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] The present invention relates to the stabilization of collagen scaffolds. (Related Applications) This application claims priority to U.S. Provisional Patent Application No. 62 / 693,192, filed July 2, 2018, which is incorporated by reference in its entirety. [Background technology]

[0002]

[0001] Visual impairment caused by abnormal refractive conditions, such as refractive error, can be a serious problem for patients of all ages and can often, but not always, be treated with subtractive laser procedures. Recently, an additive technique has been developed that involves implanting a lenticule into the patient's cornea after dissecting and folding back a flap to expose the intrastromal area of ​​the cornea. The shape of the lenticule modifies the refractive power of the patient's cornea by changing the curvature of the cornea. The flap can then be placed back on top of the lenticule. In other situations, such as keratoconus, the implanted lenticule can mechanically stabilize or stabilise the abnormal stroma and slow the progression of the disease. In cases of keratoconus, stromal pockets are more frequently formed rather than flaps.

[0003]

[0002] However, several problems limit the wider acceptance of additive (lenticule) techniques. First, the availability of donor human corneas for forming lenticules is quite limited. In addition, if lenticules are obtained from non-human sources, they need to be decellularized to minimize immune reactions. Furthermore, due to the nature of lenticules (structured layers of collagen), they are fragile and require special handling. In general, decellularization makes the structure more fragile and / or alters the permeability properties, so that decellularized lenticules are more susceptible to postoperative swelling and, as a result, the proper refractive correction at the time of surgery may not be maintained intact. Therefore, there is a need for better methods to stabilize implantable lenticules and other collagen-based scaffolds.

[0004]

[0003] Collagen is present widely in the human body, for example in the intestine, veins, joints, skin, endothelium, ventricular valves, and cornea.

[0005]

[0004] However, corneal stromal collagen is unique in many respects. Besides water, collagen constitutes the major component of the corneal stroma. Other components of the stroma include glycoaminoglycans (GAGs) and proteoglycans. Viable cells constitute only about 1-4 percent of the corneal stroma. Human stroma consists of approximately 100-150 lamellae, each containing parallel collagen fibrils. Being transparent, the layers of collagen fibrils exhibit in-plane alignment with a nearly ideal distribution of spacing between layers or lamellae. No other organ contains collagen fibrils organized in this way.

[0006] Parenchymal collagen exhibits high tensile strength in the in-plane direction, but is relatively brittle in the direction perpendicular to the plane. Decellularized parenchyma exhibits even more brittleness. Better ways to increase the mechanical strength of such decellularized parenchymal collagen are needed if it is to be used as a scaffold. Summary of the Invention [Means for solving the problem]

[0007]

[0006] A method for stabilizing a collagen scaffold is disclosed, which can use water extraction and / or compression and cross-linking to shape the collagen scaffold, mechanically strengthen the collagen scaffold, and prevent the tendency of the collagen scaffold to swell in an aqueous environment. The method of the present invention can be particularly useful for preparing collagen scaffolds as lenticules for intrastromal or intracorneal implantation as part of additive refractive surgery.

[0008]

[0007] Scaffolds formed from collagenous tissue can provide mechanical advantages to the cornea. However, the scaffolds are usually mechanically fragile due to the decellularization process and need to be strengthened and oriented before being placed in the stroma. In one aspect of the present invention, a method for forming and strengthening a scaffold from a donor collagenous tissue is disclosed, which includes the steps of: extracting a portion of tissue from a central region of a donor collagenous tissue source (e.g., donor corneal stroma); shaping the tissue portion to form a scaffold of a first desired shape; decellularizing the scaffold; compacting the scaffold (e.g., in a direction generally perpendicular to the lamellar structure of the scaffold) to remove excess fluid present in the scaffold and to increase collagen density; and crosslinking at least a portion of the scaffold to mechanically strengthen it and prevent subsequent swelling when the scaffold is exposed to an aqueous environment. Thus, the method of the present invention can enhance the mechanical strength and chemical stability of the scaffold. Where the scaffold is intended for use as an intracorneal implantable lenticule, crosslinking can be used to restore and / or ensure the optical clarity (eg, transparency) of the scaffold.

[0009]

[0008] In certain embodiments, the extracting and shaping steps can be performed simultaneously. The step of decellularizing the scaffold can further include lysing the cells, removing cellular debris from the lenticule with a detergent or surfactant, and optionally enzymatically removing at least one immunogenic epitope from the lenticule. One solution useful in decellularizing the scaffold can include water, ethyl alcohol, and glycerol. The orientation of the scaffold can also be manipulated. The method steps described herein can be performed in any order, if feasible.

[0010]

[0009] Without being bound to any particular theory of operation, the mechanical strengthening of collagen scaffolds is not only the result of crosslink formation, but also of the compaction of collagen fibrils into tighter bundles. Crosslink formation of densified fibril bundles can result in material strength improvements beyond those achievable by crosslink formation alone.

[0011]

[0010] In the method of the present invention, the crosslinking step can further include exposing at least a portion of the compressed scaffold to a crosslinking agent or an energy transfer agent, or exposing at least a portion of the compressed scaffold to radiation to induce crosslinking by peptide bond formation between collagen fibrils with or without the assistance of an energy transfer agent. A combination of chemical crosslinking and photocrosslinking is also disclosed, whereby a chemical agent can induce different chemical bonds to physically form mechanical bridges between collagen fibrils.

[0012]

[0011] The crosslinking step can be performed by exposing at least a portion of the compressed (or dehydrated) scaffold to radiation, either by direct exposure, or by exposure to such radiation at a line-of-sight angle, or via an evanescent waveguide connected to the surface of the scaffold. The crosslinking step can further include exposing a surface portion of the compressed scaffold to radiation, such that the surface portion exhibits a greater degree of crosslinking and higher collagen density than the bulk region of the scaffold.

[0013]

[0012] Preferably, the compressing and crosslinking steps result in at least a portion of the scaffold having a greater collagen density than the initial decellularized scaffold segment. For example, at least a portion of the compressed and crosslinked scaffold can have a composition of at least 20 percent collagen, preferably greater than 35 percent collagen.

[0014]

[0013] The method of the present invention can be practiced such that the scaffold is configured for use as an implantable intracorneal lenticule having a lenticule body, an oriented anterior and posterior surface, the method of the present invention further comprising treating at least a portion of the posterior surface of the lenticule with a cross-linking agent or by selective application of patterning radiation to promote attachment of the lenticule to the stroma bed. In certain embodiments, the method of the present invention can include forming a lenticule from a donor stroma, removing a portion of tissue from a central region of the donor stroma by lenticule extraction, shaping the removed tissue portion into a lenticule of a first desired shape, the lenticule having a lenticule body, an anterior surface and a posterior surface, removing cellular material from the lenticule; removing excess fluid present in the lenticule (e.g., water originally present in the stroma and any other fluids that may have been introduced into the lenticule during decellularization); and cross-linking at least a portion of the lenticule to define a final desired shape and prevent subsequent swelling when the lenticule is exposed to an aqueous environment. The formation of the lenticule can be achieved by excision or extraction of the lenticule, performed with a keratome or a femtosecond laser or an excimer laser or a water jet.

[0015]

[0014] Crosslinking of the lenticles can be achieved by exposing at least a portion of the compressed scaffold to a crosslinking agent or energy transfer agent, or by exposing at least a portion of the compressed scaffold to radiation to induce crosslinking by peptide bond formation between collagen fibrils, with or without the assistance of an energy transfer agent. For example, crosslinking of the lenticles can include exposing at least a portion of the compressed scaffold to radiation by direct exposure, or by exposure at a line-of-sight angle, or through an evanescent waveguide connected to the surface of the scaffold. In certain applications, ultraviolet radiation is a preferred energy source.

[0016]

[0015] Chemical methods have been reported for collagen cross-linking. These chemical methods can include the use of chemical agents (e.g., genipin) or photochemical agents. For example, US Patent Application Publication No. 2017 / 0119928 discloses a method for forming an artificial heart valve by decellularizing pericardial tissue to obtain a collagenous matrix material that can be cross-linked with a glutaraldehyde-containing solution and molded into a desired structure for implantation.

[0017]

[0016] Radiation crosslinking can be advantageous over chemical crosslinking because the resulting lenticules are frequently more transparent, resulting in better vision after implantation into the recipient's cornea.

[0018] Radiation crosslinking of compressed scaffolds can be achieved with substantially less energy than that required for uncompressed (fluid-laden) scaffolds. For example, for a typical lenticule after compression, crosslinking can be achieved with approximately 15 Joules / cm 2 Less than, or in some cases, about 2500 Joules / cm 2 This can be accomplished with ultraviolet radiation at a fluence of less than about 15 Joules / cm. More typically, the desired fluence is about 15 Joules / cm. 2 ~ approx. 600 Joules / cm 2 to about 280 nm. One particular range of wavelengths useful for crosslinking collagenous scaffolds generally corresponds to a portion of the "UV-C" wavelength band, e.g., from about 185 nm to about 280 nm. Other UV wavelength bands (UV-B band ranging from about 280 nm to about 315 nm (or 320 nm), or UV-A band from about 315 nm (or 320 nm) to about 400 nm) can also be used in some cases, as well as x-rays, gamma radiation, or electron beams, to induce at least partial crosslinking and / or sterilization of the scaffold.

[0019]

[0018] Because the decellularized collagen scaffold exhibits some degree of "shape memory" or "stress hysteresis," it is possible to separately compress the decellularized collagen scaffold and then crosslink it, while it is no longer constrained by the compression mold. Such a separate (continuous) manufacturing process is an alternative. However, the preferred method is in most cases to induce crosslinking while the scaffold is compressed in the mold. In either case, crosslinking of compacted collagen bundles provides greater strength than crosslinking of dispersed collagen fibril configurations.

[0020] In another aspect of the method of the present invention, the crosslinking step can further include exposing a surface portion of the compacted scaffold to radiation such that the surface portion exhibits a greater degree of crosslinking and a higher collagen density than the bulk region of the scaffold. Additionally, the crosslinking step can also include applying sufficient radiation to inactivate any microbial agents and sterilize the lenticule. In certain embodiments, the decellularizing scaffold can include removing cellular debris from the lenticule with a detergent or surfactant; and optionally further includes enzymatically removing at least one immunogenic epitope from the lenticule.

[0021]

[0020] Additionally, the compressing and crosslinking steps can result in at least a portion of the scaffold having a greater collagen density than the initial collagenous tissue segment. The crosslinking step can further include selectively applying radiation to the anterior surface such that the anterior surface region exhibits a greater degree of crosslinking or greater collagen density than a bulk region of the lenticule body.

[0022]

[0021] In yet another aspect of the present invention, a decellularized collagen lenticle is disclosed having a lenticle body derived from donor tissue with anterior and posterior surfaces formed to provide the lenticle with a desired shape and orientation. For example, the lenticle can have convex and concave surfaces that frequently, but not always, coincide with the anterior and posterior surfaces of the lenticle. The lenticle body can include a layer of collagen that has been decellularized and compressed to obtain a composition that is greater than 15 or 25 percent collagen, and can further be at least partially crosslinked to prevent axial swelling. For bulk crosslinked lenticles, the composition can be greater than 30 percent collagen. Furthermore, if a locally densified layer is desired (e.g., to approximate corneal structures such as Bowman's membrane), the local collagen concentration can be even higher (e.g., greater than 35 percent collagen, or greater than 40 percent collagen, or even greater than 60 percent collagen).

[0023]

[0022] The lenticule may, in certain embodiments, be characterized by a layer of collagen that has been cross-linked by application of radiation, and the lenticule may also be further characterized by induced peptide bonds between collagen fibrils. The lenticule may be 90 percent to 100 percent, or preferably 95 percent to 99.99 percent, free of cellular material, and following decellularization, compaction and cross-linking, the lenticule may be formed in a desired shape so that it can be implanted into a patient's eye to alter the refractive power of the cornea and replenish or strengthen areas of stromal damage or disease.

[0024]

[0023] The lenticles according to the invention typically have a curved, disc-like shape and a diameter of about 0.5 mm to about 10 mm. The lenticles also typically have a maximum thickness which may range from about 600 to about 50 micrometers, more preferably from about 400 to about 100 micrometers. The lenticles are typically not of uniform thickness, and the minimum thickness may be less than about 50 micrometers, more preferably less than about 30 micrometers or less than about 15 micrometers.

[0025]

[0024] The lenticule should exhibit low immune reactivity due to degradation of immunogenic epitopes. In certain embodiments, at least one surface of the lenticule further comprises a pattern of variable cross-linking to promote attachment of the lenticule to the stromal bed when implanted intrastromally into the stromal bed of a patient. The lenticule can also have a front surface with a front surface region having a collagen density greater than the bulk region of the lenticule body. For example, the collagen density of the front surface region can be at least about 35 percent, 40 percent, or 60 percent collagen.

[0026]

[0025] Thus, the present invention discloses a method for stabilizing the shape of collagen lenticles and scaffolds. The collagenous tissue can be harvested from any collagenous source of human or animal origin. In certain preferred embodiments, the tissue can be harvested from human or porcine parenchyma. The source tissue can be shaped during harvest or in a separate step after harvest. The tissue can be shaped, decellularized, and stabilized by various techniques, some of which are described in more detail below. Following shaping and decellularization, the harvested collagenous tissue segments are subjected to compression to reduce the fluid content and irradiated to induce crosslinking of the collagen chains or collagen fibrils. Crosslinking can be induced with or without chemical mediators, e.g., crosslinkers. In certain preferred embodiments, crosslinking is obtained by the formation of peptide bonds between collagen fibrils by exposure to sufficiently high energy radiation. This energy radiation is desirable since the creation of peptide bonds between collagen chains is usually an endothermic reaction. Various energy sources can be used to induce crosslinking of peptide bonds. In certain preferred embodiments, the energy is delivered by ultraviolet (UV) radiation.

[0027]

[0026] The present invention is particularly applicable to the spatial stabilization of collagen scaffolds harvested from lamellar collagenous tissues in general, and corneal stroma in particular. The lamellar collagenous organization of the source tissue may have evolved in nature for optical transparency, but may result in mechanical fragility and / or a permeable tendency to swell when exposed to an aqueous environment. It is therefore an object of the present invention to prevent such swelling and / or provide greater axial mechanical strength to the final scaffold. The present invention may also be applied to non-lamellar (or disorganized) excised collagenous tissue segments.

[0028] In another aspect of the invention, a method is disclosed for modifying at least one surface of the scaffold, for example the collagen density or smoothness of the anterior surface, when the scaffold is a lenticule for a corneal transplant. Such surface modification can make the scaffold easier to manipulate after implantation, for example if the overlying flap needs to be re-opened to access the implant. Surface modification can be achieved by irradiation and / or application of chemical agents.

[0029]

[0028] Decellularized and shaped corneal tissue lenticules from allograft and / or xenograft sources and methods for obtaining such lenticules are disclosed. The lenticules are particularly useful as intrastromal or intracorneal lenticule implants in keratoplasty, where a hinged flap is formed in the patient's cornea and folded back along the hinge to expose the corneal stromal bed. The shaped lenticule is then applied to the stromal bed and the flap is returned to its original position, thereby imparting a new curvature to the cornea and providing the desired refractive correction. Fine tuning of the new refractive power can be performed by laser ablation, either simultaneously with implantation or at a later time in the event of regression or tonic changes occurring.

[0030] In one aspect of the present invention, a decellularized corneal lenticule and a method for decellularizing corneal tissue are disclosed to reduce potential immunogenic responses on the part of the patient to the transplanted lenticule. Only about 1-4 percent of a normal cornea is composed of cells. The other 96-99 percent is mostly extracellular matrix (ECM), i.e., mainly collagen, glycoaminoglycans (GAGs) and proteoglycans, as well as water. In a preferred embodiment, the cellular components of the lenticule are removed, for example, by treatment with a detergent such as sodium tetradecyl sulfate (STS) or by enzymatic solubilization. If desired, particularly when the source is a non-human (xenogeneic) donor, additional steps can be taken to further reduce the immunogenicity of the lenticule. For example, two non-human epitopes that can be present in xenogeneic tissue are neu5GC and alpha-Gal. These undesirable epitopes can be present inside or on the surface of parenchymal cells; some epitopes may be buried inside GAGs, also known as mucopolysaccharides, that surround ECM collagen fibrils. In such cases, such epitopes can be selectively removed, at least in part, by kinase treatment and further washing.

[0031]

[0030] The decellularized lenticules of the present invention typically have 95%-100% of the cellular material removed. Preferably, the lenticules are 95%-99.99% decellularized. Without reciting every possible subrange between 95%-100%, it is clear that all such subranges are contemplated and considered as part of the present invention. For example, the lenticules may have 95%-97%, 97%-99%, or 98%-99.9% decellularized.

[0032]

[0031] In another aspect of the invention, a disk-shaped lenticule according to the invention is obtained by cutting a disk-shaped tissue segment from a donor cornea. The tissue segment can be sliced ​​and / or further shaped or cut so that the desired shape is obtained during the slicing procedure. The cutting can be performed mechanically, for example with a microkeratome, by laser processing, for example, by photodissection with a femtosecond laser or by an excimer laser, or by a water jet. To reduce the possibility of asymmetry, the tissue segment is preferably taken from the central part of the donor cornea, for example, with the optical or geometric axis of the donor cornea being ensured in the center of the lenticule. The shape of the tissue segment will be determined by the change in dioptric power required to correct the refractive error of the patient. For example, in the case of correction of hyperopia (hypermetropia) and / or presbyopia, the goal is usually to increase the curvature of the cornea, and the desired lenticule shape will be slightly convex on at least one side. Usually, the maximum thickness of the lenticle is less than 400 micrometers, or often less than 200 micrometers, or less than 100 micrometers, or less than 50 micrometers. For almost all applications, the maximum thickness is less than 600 micrometers. In some circumstances, vision improvement in patients suffering from macular degeneration can be achieved when the disc-shaped lenticle is formed as a prism that redirects light to different parts of the retina.

[0033]

[0032] The treatment of keratoconus can involve not only the reduction of visual aberrations but also the mechanical strengthening of the pathological stroma. For this purpose, a co-planar disk with a thickness between 50 and 300 micrometers and limited refractive influence with a transitional slope (wedge-like) zone at the periphery of the disk may be preferred. Alternatively, a lenticule for the treatment of keratoconus can take advantage of the natural curvature inherited from the donor cornea or can introduce an additional degree of curvature during compression and / or cross-linking by using a curved mold. In some cases, it may be advantageous to transplant the lenticule "upside down" in treating keratoconus, for example, so that the curvature of the lenticule is the opposite of that of the recipient cornea.

[0034]

[0033] In some cases, a curved mold is desirable, but the compression and cross-linking steps can also be performed in a flat mold. (A flat mold can be advantageous for storage and transportation, or efficiency of manufacturing.) In another aspect of the invention, a sealed mold can be used for both compression and storage.

[0035] Pressure applied to one surface of the scaffold can also be used to change the curvature of the surface and / or realign (or maintain) the collagen fibrils. For example, the scaffold can be fixed to the opening of a chamber, which is then filled with a pressurized fluid to apply pressure on one side of the scaffold, thus exerting a horizontal / tangential force on the scaffold. The pressure of the fluid will cause the scaffold to expand (like a balloon). A compressible plate can optionally be applied to the opposite surface. For example, a curved (quasi-concave) compression plate can be used to limit the extent to which the scaffold can be stretched or reshaped. Once the desired curvature is obtained, cross-linking can be used to maintain the desired shape and / or prevent the tendency of such engineered scaffolds to swell upon intrastromal or intracorneal implantation.

[0036]

[0035] In some embodiments, it may be advantageous to ensure that the upper stromal surface, i.e., the so-called "Bowman's membrane", so that the anterior surface of the lenticule will exhibit a different texture than the other (posterior) surface, since the natural anterior segment of the upper stromal surface of the cornea is denser and smoother due to the natural compaction of the outermost layer of stromal tissue. Alternatively, the excised segment may be taken from the central region of the stroma, and the anterior surface may be densified following shaping and excision by selective cross-linking as described in more detail below. The posterior surface (opposite the anterior surface or Bowman's membrane) will be rougher due to the lower density of the stromal tissue and the fact that it is formed by mechanical or laser cutting of the tissue. This difference in roughness may be particularly advantageous when the lenticule is used for intrastromal or intracorneal implantation, since it may be highly desirable for the lenticule to adhere strongly to the stromal bed. If suboptimal refractive outcomes are observed after the procedure, it may be necessary to re-fold the flap to allow for further corneal implantation (re-sculpting of the lenticule), such as by laser ablation. Any displacement of the lenticule from its original position in the stromal bed may compromise the efficacy of such a corneal implantation. Furthermore, the smoothness of the anterior surface of the lenticule also reduces the chance that re-dissecting the flap will result in the lenticule being dislodged.

[0037] In yet another aspect of the invention, the posterior surface can be treated after harvesting, shaping, and decellularization to make it more adherent to the stromal bed. For example, a crosslinking agent can be applied prior to sterilization and packaging. Alternatively, an attachment enhancer can be applied by the clinician during the procedure prior to implantation. The anterior surface can be treated to make it less likely to adhere to the flap. [Brief description of the drawings]

[0038] [Figure 1A] FIG. 1 is a schematic cross-sectional view of an excised parenchymal tissue segment. [Figure 1B]FIG. 2 is a schematic cross-sectional view of an excised parenchymal tissue segment after decellularization, showing tissue swelling and resulting spacing of collagen fibrils. [Figure 1C] FIG. 1 is a schematic cross-sectional view of an excised parenchymal tissue segment after decellularization, compression and cross-linking in accordance with the present invention. [Diagram 2] FIG. 1 is a schematic cross-sectional view of an excised parenchymal tissue segment following decellularization of the anterior region, compression and selective cross-linking. [Diagram 3] FIG. 1 is a schematic cross-sectional view of an excised parenchymal tissue segment following decellularization, compression and selective patterning of the posterior region. [Figure 4A] FIG. 2 illustrates a lenticule according to the present disclosure in a flat configuration, which is a typical configuration during manufacturing and / or shipping. [Figure 4B] FIG. 1 illustrates a lenticule in its final curved state prepared for intracorneal implantation. [Figure 5A] FIG. 1 illustrates a lenticule designed for intracorneal implantation to correct a hyperopic condition. [Figure 5B] FIG. 1 illustrates a lenticule designed for intracorneal implantation to correct a myopic condition. [Figure 5C] FIG. 1 illustrates a lenticule designed for intracorneal implantation to correct a presbyopic condition. [Figure 5D] FIG. 1 illustrates a lenticule designed for intracorneal implantation to correct the condition known as keratoconus. [Figure 6A] FIG. 13 illustrates another embodiment of a lenticule fabricated with localized spots of strong cross-linking. [Figure 6B] FIG. 13 illustrates yet another embodiment of a lenticle that includes a central optically active zone with moderate crosslinking and an outer or peripheral zone with strong crosslinking. [Figure 7A] FIG. 1 illustrates the use of a lenticule for deep lamellar keratoplasty (DALK). [Figure 7B]1A-1C illustrate the use of a lenticule configured for placement over an intact Bowman's membrane of a patient. [Figure 7C] FIG. 1 illustrates yet another embodiment of a lenticule for penetrating keratoplasty (PK) surgery. [Figure 8A] Figures 8A and 8B illustrate two alternative designs for the periphery of a thick lenticule: Figure 8A shows a lenticule with a simple, e.g. cylindrical or conical, periphery. [Figure 8B] Figures 8A and 8B illustrate two alternative designs for the periphery of a thick lenticle: Figure 8B shows a lenticle with a zigzag or stepped edge on the periphery of the lenticle; [Figure 8C] FIG. 1 shows a lenticule having a "hook" edge around the periphery of the lenticule. [Figure 9] FIG. 1 is a schematic perspective view of a pressing device for use in the steps of compressing and crosslinking the collagen scaffold according to the present invention. [Figure 10A] Figures 10A and 10B illustrate a two-part sealed compression and storage mold according to the present invention: Figure 10A shows the mold prior to compression of the scaffold. [Figure 10B] Figures 10A and 10B illustrate a two-part sealed compression and storage mold according to the present invention, with Figure 10B illustrating the mold after compression. [Figure 11] FIG. 14 illustrates a further device according to the invention for extending a scaffold. [Figure 12] FIG. 12 shows yet another alternative device similar to that of FIG. 11 but with the addition of a compression-applying plate element for simultaneous scaffold stretching and scaffold compression, as well as to facilitate exposure to crosslinking radiation. [Figure 13] FIG. 1 illustrates an apparatus 130 for measuring the transparency of lenticules made in accordance with the present disclosure. [Figure 14] 14 is a graph of a luminance distribution curve obtained with an apparatus such as that of FIG. 13 for quantifying optical transparency. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039]

[0065] The present invention is not limited to the specific process, composition, or methodology described, since the process, composition, or methodology may vary. The terms used herein are only for the purpose of describing a particular version or embodiment of the present invention, and are not intended to limit the scope of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All publications mentioned herein are incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0040]

[0066] The term "dissect" encompasses any of the known methods of dissection, ablation or removal of biological material, for example, by the action of a mechanical blade, an ultraviolet (UV) laser, a femtosecond laser, or a water jet.

[0041]

[0067] The term "compression" includes compaction by application of pressure or by other techniques such as vacuum or centrifugally driven water extraction.

[0042]

[0068] The term "radiation" includes infrared radiation, visible radiation, and ultraviolet radiation (eg, from about 400 nm down to approximately 193 nm or below), X-rays, gamma rays, and electron beam.

[0043]

[0069] The term "biological sample" refers to a tissue, a cell, a cell extract, a homogenized tissue extract, or a mixture of one or more cellular products. A biological sample can be used or present in a suitable physiologically acceptable carrier.

[0044]

[0070] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to a "cell" is a reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth.

[0045]

[0071] As used herein, the term "about" means plus or minus 10% of the numerical value of the number it is used with. Thus, about 100 μm means a range of 90 μm to 110 μm.

[0046]

[0072] The terms "animal," "patient," or "subject" as used herein include, but are not limited to, humans and non-human vertebrates, such as wild, domestic, and captive animals. The terms "animal," "patient," or "subject" also refer to the recipient of a corneal lenticule transplant. The term "xenograft" refers to tissue collected from donor animals, including pigs (porcine), cows, apes, monkeys, baboons, other primates, and any other non-human animals. An "allograft" refers to tissue taken from a donor that is the same species as the recipient.

[0047]

[0073] In general, the term "tissue" refers to any collection of similarly specialized cells that are united in the performance of a particular function. The corneal stroma, which is mostly non-cellular (approximately 1-5% is cellular), is an example of a "tissue."

[0048]

[0074] The term "lenticule" refers to decellularized, processed donor corneal tissue ready for transplantation into or onto a recipient's cornea. Unless otherwise specified, the terms "lenticule" and "scaffold" are used interchangeably herein. The terms "collagen concentration" and "collagen percentage" are used interchangeably herein and refer to the amount of collagen present in a lenticule or scaffold. This concentration or percentage can be measured as the weight of the fraction of a completely dried lenticule, e.g., a vacuum-dried lenticule, relative to the weight of the lenticule before drying (which is in perfect equilibrium with moisture). In some cases, ethanol can be used to improve drying.

[0049]

[0075] The term "intracorneal" in the context of lenticule transplantation refers to any procedure in which a lenticule is placed in or on the cornea. One type of intracorneal transplantation is an "intracerebro" transplantation, which is a procedure in which a lenticule is placed in the stroma of the eye without removing the anterior Bowman's membrane or epithelium, for example, by turning back a flap of anterior tissue or by direct insertion via a lateral approach. Other types of intracorneal uses of lenticules include deep lamellar keratoplasty (DALK) and penetrating keratoplasty (PK), in which the lenticule completely replaces the anterior segment of the eye, as discussed in more detail below. Yet another applicable "intracorneal" procedure is the so-called "lamellar keratoplasty", which is also discussed in more detail below.

[0050]

[0076] The term "axial" refers to the direction relative to the orientation of the lenticule or scaffold. Typically, a shaped lenticule is curved in a spheroidal or ellipsoidal disc-like shape, with the axial direction or "axis" generally perpendicular to the center of the disc. Unless otherwise specified, the "axial" is also generally nearly parallel or coaxial with the visual or optical axis of the eye from which the tissue segment is harvested or the recipient eye into which the lenticule is designed to be implanted. (In natural eyes, the optical axis usually passes nearly, but not exactly, through the center of the cornea.)

[0051]

[0077] Planetary physics (spherical geometry) can provide a convenient nomenclature to describe the spatial organization of collagen fibrils in the stroma. The majority of collagen fibrils are in high to mid-latitude orbits, with only a small portion in polar orbits. For this reason, the stroma is usually thinnest in the central (middle) region / location. The axial direction of compression can be referred to as polar compression or radial compression. The central region of the donor stroma exhibits strong rotational symmetry in fibril alignment. If the implant is specified to function under tensile stress as well as in compression, lenticules extracted from the central stroma location would preferably impart rotationally symmetric tensile properties to the implant.

[0052]

[0078] The present disclosure also relates to decellularized corneal lenticules from allograft and / or xenograft sources and methods of forming lenticules from donor stroma. The decellularized corneal lenticules of the present disclosure can be used to correct abnormal refractive conditions such as myopia, hyperopia, presbyopia, and astigmatism, as well as other ophthalmic conditions.

[0053]

[0079] The cornea can generally be considered to be composed of five layers from anterior to posterior: the corneal epithelium, a thin but dense upper stromal layer (usually called Bowman's membrane in the human eye), the corneal stroma, Descemet's membrane, and the corneal endothelium. The corneal epithelium is composed of about six layers of non-keratinized stratified squamous epithelial cells, which are fast-growing and easily regenerate. The anterior stromal layer (e.g., Bowman's membrane) is a tough layer composed of mostly randomly organized, tightly interwoven collagen type I fibrils. The corneal stroma is a thick, transparent layer composed of collagen type I fibers organized in parallel layers. Descemet's membrane is a thin, non-cellular layer that serves as the basement membrane of the corneal endothelium and is composed of low-rigidity collagen type IV fibrils. Finally, the corneal endothelium is composed of a single flat or low cubic monolayer of mitochondria-rich cells.

[0054]

[0080] As used herein, the term "Bowman's membrane" is used to describe any anterior stromal region of the cornea, either from a human or donor animal cornea. Although the layer of densification in the human cornea may be more pronounced (hence known as Bowman's membrane in the human cornea), all corneas exhibit somewhat greater anterior densification and smoothness (relative to the stromal floor tissue) to some degree depending on the species and age of the animal. Thus, "Bowman's membrane" is the term used throughout this application to describe such anterior segment.

[0055]

[0081] Therefore, the harvesting and processing of the donor cornea and the creation of the lenticule are very important factors in the correction of refractive errors of vision. The lenticule is particularly useful as a lenticule implant in corneal transplantation, where a hinge-like flap is formed in the patient's cornea and folded back along the hinge to expose the stromal bed of the cornea. A shaped lenticule is then applied to the stromal bed and the flap is returned to its original position, resulting in a new curvature of the cornea and the desired refractive correction. Fine tuning of the new refractive power can be performed by laser ablation, either simultaneously with the transplantation or at a later time in the case of regression or tonic changes occurring.

[0056]

[0082] In certain embodiments of the present invention, the decellularized corneal lenticule can include a lenticule body derived from donor stroma, having an anterior surface including at least a portion of the top layer from the donor stroma, and a posterior surface formed to provide a lenticule with a desired shape; wherein the donor stroma is decellularized. In other embodiments, the lenticule is shaped without regard to retaining Bowman's membrane and any upper portion of the donor stroma. For example, the lenticule can be formed by intrastromal extraction of a stromal tissue segment, for example, by extraction with femtosecond laser pulses.

[0057]

[0083] In certain embodiments, donor stroma is harvested and decellularized to produce a lenticule with reduced potential of any immunogenic response on the part of the patient. Only about 1 to about 4 percent of a normal cornea is composed of cells. The other 96 to 99 percent is mostly extracellular matrix (ECM), primarily collagen, as well as water, glycoaminoglycans, and proteoglycans. As noted above, the decellularized lenticules of the present invention are typically free of 95% to 100% of cellular material. Preferably, the lenticules are 95% to 99.99% free of cells. Without reciting every possible subrange between 95% and 100%, it is clear that all such subranges are contemplated and considered as part of the present invention. For example, the lenticules may be 95% to 97%, 97% to 99%, or 98% to 99.9% free of cellular material. The amount of cellular material remaining in the lenticule can be assessed, for example, by residual DNA or RNA content. Preferably, the DNA or RNA content is less than 1 weight percent, or less than 0.1 weight percent, or less than 0.01 weight percent of the original DNA or RNA content.

[0058]

[0084] Decellularization, or removal of cellular material from the donor stroma, can be accomplished using a variety of techniques. In a preferred embodiment, the corneal cellular material is removed by chemical treatment. Chemicals used to dissolve and remove cells from the cornea include detergents such as sodium tetradecyl sulfate (STS), acids, alkaline treatments, ionic detergents such as sodium dodecyl sulfate (SDS), non-ionic detergents such as Triton X-100, and zwitterionic detergents. In some embodiments, the corneal cellular material is removed using enzymatic treatment. Lipases, thermolysins, galactosidases, nucleases, trypsins, endonucleases, and exonucleases are used to remove cellular material from the cornea. In some embodiments, the corneal cellular material is removed using physical techniques. These physical techniques include methods used to dissolve, kill, and remove cells from the tissue matrix through the use of temperature, force, and pressure, as well as electrical disruption. Temperature methods are often used with a rapid freeze-thaw mechanism. Temperature methods preserve the physical structure of the ECM scaffold. Pressure decellularization involves the controlled use of hydrostatic pressure at elevated temperatures to avoid unsupervised ice crystal formation that could damage the scaffold. Electrical disruption of the plasma membrane is another option for dissolving the cellular material of the cornea.

[0059]

[0085] In the embodiments described herein, the lenticules can exhibit even lower immunoreactivity due to degradation of immunogenic epitopes. This can be an important step when using xenogeneic donation. For example, two non-human epitopes that can be present in xenogeneic tissues are N-glycolylneuraminic acid (Neu5GC) and galactose-alpha-1,3-galactose (alpha-Gal). These undesirable epitopes can be present not only inside but also on the surface of keratocytes; a minority of epitopes may be buried in glycoaminoglycans (GAGs), also known as mucopolysaccharides, that surround ECM collagen fibrils. In certain embodiments, epitopes can be selectively removed by enzymatic treatment, such as by galactosidase treatment, and further washing. Alternatively, corneal tissue can be harvested from knockout transgenic animals (e.g., transgenic pigs) that lack any immunogenic epitopes, thus making it possible to create non-immunogenic lenticules without the need for an epitope degradation step.

[0060]

[0086] In the embodiments described herein, the decellularized lenticule can be further sterilized in conjunction with packaging and sealing. Sterilization can be performed using a wet agent, radiation, or electron beam. In a preferred embodiment, sterilization of the decellularized lenticule is performed using UV radiation, as damage to the collagen scaffold is less likely to occur. The use of UV radiation can be advantageous in improving the optical transparency of the lenticule.

[0061]

[0087] In another aspect of the invention, the shape and orientation of the lenticules are designed for optimal results. In some embodiments, the diameter of the lenticules is from about 0.5 millimeters (mm) to about 10 mm, or from about 3 mm to about 9 mm, or from about 4 mm to about 8 mm, or from about 5 mm to about 7 mm. Again, without reciting every possible subrange between 0.5 mm and 10 mm, it is clear that all such subranges are contemplated and considered as part of the present invention.

[0062]

[0088] The donor corneal stroma can be sliced ​​and / or further shaped to obtain the desired shape. The slicing can be performed mechanically, for example, using a microkeratome or the like, or by laser processing, for example, by photoablation using an excimer laser or photodissection using a femtosecond laser. To reduce the possibility of asymmetry, the corneal tissue segment is preferably taken from the central part of the donor cornea, for example, with the optical or geometric axis of the donor cornea being ensured in the center of the lenticule. The shape of the corneal tissue segment will be determined by the diopter change required to correct the refractive error of the patient. For example, in the case of correction of hyperopia (farsightedness) and / or presbyopia, the goal is usually to increase the curvature of the cornea, and the desired lenticule shape will be slightly convex on at least one side. In some embodiments, the maximum thickness of the lenticule will be less than 600 micrometers, less than 400 micrometers, less than 200 micrometers, less than 100 micrometers, or less than 50 micrometers. The smaller the diameter and the thinner the lenticule, the faster the lenticule will integrate into the patient's stromal bed.

[0063]

[0089] Stromal collagen fibrils are long polymer (polypeptide) strings. Stromal collagen fibrils are triply twisted proteins. The length of a single collagen fibril is nearly macroscopic, and therefore each fibril can be a strong scatterer of light individually. The fact that stroma is axially transparent is the result of the negative sum of all these strongly scattering contributions. That is, collagen fibrils collectively contribute to a collective near-zero scattering in total, despite their individual scattering. This collective transparency is obtained when the fibrils are organized parallel to one plane. In the corneal stroma, the plane of organization is perpendicular to the optical axis. This unique organization is present in the cornea, but is not observed in other organs. In other organs, such as the intestine, myocardial membrane, and ventricular valves, the fibrils are not precisely aligned, and therefore light is scattered. The same is true for the limbal collagen of the eye.

[0064]

[0090] In addition to transparency, natural selection has optimized the corneal structure for strength as well. The length and alignment of collagen fibrils contribute substantially to the in-plane (tangential) tensile strength of the stroma. The natural eye shape is maintained by hydrostatic intraocular pressure, which exerts a tangential tensile stress on the stroma. The in-plane orientation of collagen fibrils contributes to corneal transparency and provides significant mechanical tensile strength. However, this strength is mostly limited to forces applied in-plane. In comparison, the strength of the corneal stroma along the optical axis is substantially weak. One manifestation of this lack of axial strength is the swelling of the scaffold when immersed in water, e.g., buffered saline solution (BSS). Swelling is almost exclusively unidirectional, in the direction of the optical axis. Swelling in the in-plane direction is negligible.

[0065]

[0091] This fragility of the scaffold is a source of concern since the form fit of the optically active lenticule is crucial for successful additive refractive surgery: Swelling of the scaffold in the axial direction can induce refractive error.

[0066]

[0092] The amount of swelling is often correlated with the immersion liquid. The highest degree of swelling is observed when surfactants and / or detergents are added to the water, i.e., approximately 250-400% of the nominal thickness of the scaffold. BSS usually induces swelling of approximately 150-250% of the nominal thickness. Alcohols (both light and heavy) usually cause less swelling. (Nominal thickness can be defined as the axial thickness of the original excised parenchymal segment (lamella) before the decellularization step, e.g., less than about 60 seconds or thus after excision, e.g., if the tissue specimen is very fresh, or it can be longer if the specimen retains epithelial and / or endothelial cells).

[0067]

[0093] In the case of thick lenticules, swelling can cause the lenticule to become so thick that it becomes difficult to place the flap back onto the stromal bed. The flap may also be too short to cover the added material (increasing the risk of epithelial undergrowth) and may require further steps of mechanical stretching and / or suturing of the flap to the stromal bed.

[0068]

[0094] The unidirectional mechanical fragility of the collagen scaffold, together with changes in the permeability properties and / or water affinity of the scaffold (compared to the stroma before decellularization), can lead to significant unidirectional swelling in the direction of the thickness of the scaffold (i.e., in the axial direction).

[0069]

[0095] Free water exists within collagenous tissue. In intact natural corneas, the water content of the stroma is controlled by the overall structure of the cornea, such as the epithelial and endothelial membranes that form the border of the cornea. However, when exogenous stromal tissue is transplanted during additive refractive surgery, this balance is often disturbed, and the water content of the transplanted lenticule tends to increase and cause postoperative swelling beyond the nominal thickness.

[0070]

[0096] According to the invention, placing a flat (cylindrical) collagenous tissue specimen between two plates that apply constant pressure can reduce the fluid content (or, equivalently, increase the collagen concentration). The same applies to shaped lenticules that have undergone decellularization. The press must provide drainage so that excess free fluid can be drawn off laterally from the tissue. In the case of shaped lenticules, at least one of the plates of the press must be curved to accommodate the radial changes in the thickness of the lenticule. Preferably, pressure is applied gently for the desired time. Depending on the desired degree of compression, pressure can be applied to the collagen scaffold for a predetermined period ranging from a few seconds to several hours, for example from 30 seconds to 1 hour, or in some cases from 5 minutes to 30 minutes.

[0071]

[0097] The distance traveled by the plates of the press allows the calculation of the approximate collagen concentration. For example, if a flat excised tissue segment has a nominal thickness of 100 micrometers and the segment swells to 200 micrometers after the decellularization process, the collagen content of the composition can be about 15%. If the segment were then recompressed to a thickness of 100 micrometers, the nominal collagen concentration would recover to a level of approximately 30%. If the scaffold were further compressed to a thickness of 50 micrometers, the collagen concentration would be approximately 60%. If the scaffold were further compressed to a thickness of 40 micrometers, the collagen concentration would be approximately 75%. (The collagen content percentage can be measured as the weight fraction of the vacuum-dried lenticule relative to the weight of the lenticule before drying, when the lenticule is in perfect equilibrium with water.) This value is only obtained after prolonged pressure and is close to the upper limit of collagen concentration that is reasonably achievable. The remaining water at this point is tightly bound to the protein, for example by van der Waals forces, and further pressure may compromise the integrity of the collagen fibrils.

[0072]

[0098] The process of compressing collagenous tissue is largely reversible when the scaffold is removed from the press and reimmersed in fluid. The scaffold reswells to approximate its pre-compression thickness. However, in another aspect of the invention, a method is disclosed that prevents reswelling by strengthening the collagen scaffold in the axial strain direction, most notably via cross-linking of the collagen scaffold during pressing. Compression of the scaffold and / or removal of excess fluid can also be achieved by exposing the scaffold to acceleration, e.g., centrifugation. For example, 10 G to 100 G (981 m / s 2 ) or greater may be used. Removal of excess fluid may also be accomplished or assisted by exposing the scaffold to a vacuum or reduced pressure.

[0073]

[0099] In certain embodiments, crosslinking can be achieved by chemical agents. External chemical molecules can be added (mainly in aqueous solutions or other fluids) at sufficient concentration, duration, and temperature. The molecules can be constructed to bond with one collagen fibril at one end and another collagen fibril at the other end. The type of bond can be specific to the agent or does not have to be a peptide type bond. The chemical molecules create physical bridges with chemical bonds (covalent bonds) as strong attachments. A sufficiently dense population of such bridges gives new strength and / or rigidity to the axial collagen scaffold. The collagen molecules do not have to be in contact with each other, but should be located at a distance of approximately the molecular size of the agent. This gentle actuation requirement facilitates the crosslinking process. Examples of chemical crosslinkers are glutaraldehyde, genipin, and simple sugars.

[0074]

[0100] Collagen fibrils are strong light scatterers themselves, but the orientation and statistical organization of collagen fibrils collectively eliminates scattering. A potential drawback of crosslinkers and energy mediating molecules is that they introduce exogenous substances into the collagen scaffold that can act as light scatterers and adversely affect the optical transparency of the collagen scaffold when it is used as an implantable lenticule in refractive surgery.

[0075]

[0101] In other embodiments, collagen fibrils can also be strengthened by stable chemical bonds established directly between fibrils. This occurs when fibrils are in contact with each other, but does not occur naturally. The so-called peptide bonds are endothermic and require localized and timely delivery of external energy to the location where the fibrils contact. In some embodiments, specialized intermediary molecules can be used that receive energy from light quanta. The intermediary molecule can then provide the bond energy or catalyze the crosslinking process without itself participating in the structure of the link. Thus, light is an indirect energy source for building stable bonds. An example of an intermediary molecule is riboflavin when exposed to light, such as from a light-emitting diode (LED).

[0076]

[0102] In yet another variant, the collagen fibrils of the scaffold can be directly bonded to each other by radiation that is absorbed locally where the fibrils contact each other. No exogenous intermediary molecule is required to capture the quantum of energy (although endogenous molecules such as glycoaminoglycans (GAGs) present in collagenous tissues may provide a similar function). The quantum is absorbed directly and in a timely manner at the junction of the contact event. This method can utilize various forms of direct irradiation, such as visible light, blue or UV radiation, gamma rays, or even electron beams. One preferred energy source is UV light with an energy density of at least 100 joules per square centimeter, or at least 200 joules per square centimeter, or at least 300 joules per square centimeter. For example, desirable energy densities of actinic radiation can range from about 100 to about 5000 Joules per square centimeter, or between about 200 to about 1000 Joules per square centimeter, or between about 300 to about 600 Joules per square centimeter.

[0077]

[0103] The density (compression) of the collagen scaffold can play a role in the rate of crosslink formation: if the collagen concentration is higher (i.e., if the collagen scaffold is more compressed), the process of crosslink formation can proceed faster.

[0078]

[0104] The critical (threshold) dose required for pure radiation-induced crosslink formation alone (without any mediator such as riboflavin) may be correlated with the decellularization process. The threshold dose for non-decellularized parenchyma may be 3-300 times greater than the threshold dose for decellularized collagen scaffolds. (This ratio may also be correlated with the wavelength of radiation applied.) In another aspect of the present invention, it was discovered that the collagen crosslink formation threshold is lowered when the decellularization removes more of the extracellular material (such as GAGs) present in the parenchyma. The difference in thresholds may vary by an order of magnitude depending on the decellularization protocol and the wavelength of radiation applied. This lower crosslink formation threshold appears to be correlated with the intensity of the decellularization protocol.

[0079]

[0105] Crosslinking can be carried out via a dedicated UV radiation source (or by ambient UV radiation, e.g. sunlight), either simultaneously with compression or in a subsequent step.

[0080]

[0106] When light energy is used to induce crosslinking, the absorption of light may be determined by Beer's law. That is, more light will be absorbed in the surface layer of the material where the light impinges, and less will be absorbed in the deeper layers of the material. The amount of light available to induce crosslinking essentially decays exponentially. If there is a difference in the amount of light scattering molecules in the scaffold, this may also affect the distribution of energy, since the scattering agents will reduce the amount of light that can pass to the underlying region of the irradiated material. In the present invention, such effects can be effectively used to create graded degrees of crosslinking and / or to impart different properties to the surface regions of the scaffold exposed to actinic radiation.

[0081]

[0107] Thus, the natural absorption profile of light energy alone or in conjunction with the introduction of light scattering agents (e.g., as a surface coating) provides the option of selective surface crosslinking and / or less crosslinking below the surface of the collagen scaffold. Selective crosslinking of the surface of the lenticule may have advantages in imparting different adhesiveness, permeability, or smoothness to the surface, or in facilitating greater or less penetration by recipient cells after implantation. For example, if a scaffold is compressed to about 60% collagen density and selectively crosslinked at the surface to a depth of about 5-10 micrometers, the surface will retain high density, while the remainder of the scaffold will still remain unchanged from its expanded state after the decellularization process. Such a densified surface can form a pseudo-Bowman's membrane.

[0082]

[0108] Additionally, surface crosslinking can be used to impart patterned effects on the front, back, or both surfaces by selectively treating portions of the surface with light scatterers or exposing the scaffold through a patterned mask. Surface patterning can selectively modify the friction or adhesion of portions of the surface.

[0083]

[0109] There are three ways to selectively crosslink the surface of the scaffold. First, the depth of penetration can be limited by using short wavelength UV radiation, which has strong absorption in the scaffold. In this approach, preferred wavelengths range from about 230 to about 150 nanometers, more preferably from about 215 to about 193 nanometers (e.g., 193 nm). The impact angle of the radiation is usually normal to the surface, but may vary between 0 and 60 degrees relative to the surface normal.

[0084]

[0110] Alternatively, longer wavelength UV radiation can be used, for example, up to about 400 nanometers, penetrating the scaffold at small angles of incidence, such as greater than 60 degrees, preferably greater than or equal to 75 degrees, for example, in the range of about 80 degrees to about 89.9 degrees. One advantage of this method is that reliable and powerful UV radiation sources at wavelengths of about 280 nanometers or more are readily available, since such sources are in industrial use. The use of laser radiation as a light source may also be preferred, since the spatial coherence of laser light allows for good definition of small angles of incidence.

[0085]

[0111] Yet another alternative is to use an evanescent wave slab waveguide, which allows for very shallow cross-linking depths (on the order of the wavelength of the incident light). For example, when using 380 nanometer UV radiation, cross-linking can be limited to a surface layer of less than a micrometer.

[0086]

[0112] Importantly, by densifying (or more aggressively selectively cross-linking) the anterior surface of the lenticule, the anterior surface of the lenticule will exhibit a different texture than the posterior surface, since Bowman's membrane is denser and smoother due to tightly interwoven collagen type I fibrils. The posterior surface (opposite the anterior surface) will be rougher due to the lower density of the composition of this portion of the lenticule (and the fact that it is formed by mechanical or laser cutting of the tissue). This difference in roughness can be particularly advantageous when the lenticule is used for intrastromal or intracorneal implantation, since it can be highly desirable for the lenticule to adhere firmly to the stromal bed. If suboptimal refractive results are observed after the procedure, it may be necessary to fold the flap back again to allow for further corneal implantation (recutting the lenticule), such as by laser ablation. Any displacement of the lenticule from its original position in the stromal bed can compromise the effectiveness of such a corneal implantation. Furthermore, the smoothness of the anterior surface of the lenticule also reduces the likelihood that re-cutting the flap will result in the lenticule becoming dislodged.

[0087]

[0113] In yet another aspect of the invention, the posterior surface can be treated after harvesting, shaping, and decellularization to allow the posterior surface to be more attached to the stromal bed. For example, a crosslinking agent or attachment enhancer can be applied prior to sterilization and packaging. Alternatively, a crosslinking agent or attachment enhancer can be applied by the clinician during the procedure prior to implantation.

[0088]

[0114] In some embodiments, the harvested, shaped and decellularized lenticule is marked in a manner that allows the clinician to maintain the proper orientation of the lenticule during re-dissection of the flap and re-cutting with the laser. The marking can be done in a variety of ways, but in all cases will be invisible to the patient once the lenticule is placed in the stromal bed. In some embodiments, the marking can be a microscopic incision in the top anterior portion or bottom anterior portion of the lenticule. In some embodiments, the marking can be a line or dot made with dye placed in the top anterior portion or bottom anterior portion of the lenticule.

[0089]

[0115] Also disclosed herein is a method of forming a lenticle from donor stroma. In some embodiments, the method of forming a lenticle from donor stroma includes removing a portion of stroma from a central region of the donor cornea and shaping the posterior surface of the donor stroma to form a lenticle body of a desired shape. To reduce the possibility of asymmetry, the tissue segment is preferably taken from the central portion of the donor cornea, e.g., with the optical or geometric axis of the donor cornea secured in the center of the lenticle. The shape of the tissue segment will be determined by the diopter change required to correct the patient's refractive error. For example, in the case of correction of hyperopia (farsightedness) and / or presbyopia, the goal is usually to increase the curvature of the cornea, and the desired lenticle shape will be slightly convex on at least one side. The lenticle may also include one or more asymmetric markers (such as the letter "L") on the periphery of the lenticle to identify the anterior and posterior surfaces of the lenticle.

[0090]

[0116] The scaffolds of the present invention may provide further advantages as refractive (e.g., additive) lenticules in that the scaffolds may be designed to have a higher refractive index than native stromal tissue. Currently, the use of implanted lenticules to modify the curvature of the cornea is typically limited to a total diopter power (e.g., the sum of the diopter values ​​of the natural cornea and the additive lenticule) of less than about 50D (measured near the apex) before epithelial instability or unacceptable epithelial erosion occurs. Implantable scaffolds with higher refractive indexes offer the potential for higher hyperopic correction. The standard refractive index of stromal tissue is typically about 1.376. The compression techniques of the present invention may provide scaffolds with refractive indices greater than 1.377, greater than 1.378, greater than 1.379, greater than 1.38, or higher. A gradient in the refractive index of the lenticule may also be achieved by controlled cross-linking. (Such gradients may also be utilized to correct higher order refractive errors, a term that may be described by higher order Zernike polynomials for the human eye).

[0091]

[0117] In the embodiments described herein, the method of the invention creates lenticules with a shape and density designed for optimal results. The lenticules are obtained by first slicing a disk-shaped tissue segment from the donor stroma, preserving Bowman's membrane as the anterior surface. In some embodiments, the diameter of the lenticule is from about 0.5 mm to about 10 mm, or from about 3 mm to about 9 mm, or from about 4 mm to about 8 mm, and from about 5 mm to about 7 mm. The tissue segment can be sliced ​​and / or further shaped or sliced ​​to obtain the desired shape during the slicing procedure. Slicing can be performed mechanically, for example, by laser machining, such as with a microkeratome, or by photodissection using a femtosecond laser. Slicing can be performed using instruments such as those disclosed in International Patent Application No. PCT / IB2016 / 054793, entitled "Surgical Apparatus and Blade Elements for slicing Lamellar Segments From Biological Tissue," which is incorporated herein by reference in its entirety.

[0092]

[0118] The lenticule can also be obtained by femtosecond laser ablation, excimer laser ablation, or by cutting with a water jet. If preservation of the anterior segment is not required, the lenticule can also be obtained by the Small incision Lenticule Extraction (SMILE) technique, as disclosed, for example, in U.S. Patent No. 6,110,166, entitled "Method For Corneal Laser Surgery," which is incorporated herein by reference in its entirety.

[0093]

[0119] In some embodiments described herein, the maximum thickness of the lenticle will be less than 600 micrometers, less than 400 micrometers, less than 200 micrometers, or less than 100 micrometers, or less than 50 micrometers. The smaller the diameter and the thinner the lenticle, the faster the lenticle can integrate into the patient's stromal bed.

[0094]

[0120] In certain embodiments, the donor stroma is decellularized to produce a lenticule with reduced potential for adverse patient reactions to cell-derived immunogens. The decellularized lenticule produced using the methods of the present invention is 90-100 percent, or preferably 95-99.99 percent, or 98-99.9 percent, free of cells and / or cellular debris. Only about 2 percent of a typical cornea is composed of cells. The remaining 98 percent is mostly extracellular matrix (ECM), primarily collagen, water, GAGs, and proteoglycans. The preferred and mostly practiced method of characterizing the amount of cellular debris after the decellularization process is based on the detection of DNA or alternatively RNA residues. These methods are highly sensitive and highly specific. These measurements are typically normalized to the amount of DNA / RNA present in the lenticule prior to the decellularization step. Without reciting every possible subrange between 90% and 99.99%, it is clear that all such subranges are contemplated and considered as part of the present invention. For example, the produced lenticules are 90%, 95%, 99%-99.7%, 99.7%-99.9% or better free of native cellular material (as assessed by residual DNA / RNA content). In other words, the amount of cellular material remaining in the lenticule can be less than 1 weight percent, or less than 0.1 weight percent, or less than 0.01 weight percent of the original DNA or RNA content, as assessed by residual DNA or RNA content. (By virtue of the lenticule extraction itself, a significant amount of decellularization can be performed; as much as 95% of the total corneal cell content is in the epithelium and endothelium, which can be mechanically disposed of, leaving only the corneal stroma for further decellularization.)

[0095]

[0121] Removal of cellular material from the donor stroma (decellularization) can be accomplished using a variety of techniques. In a preferred embodiment, the cellular material of the cornea is removed by chemical treatment. Chemicals used to dissolve and remove cells from the cornea can include acids, bases, detergents (e.g., sodium tetradecyl sulfate (STS)), ionic detergents (e.g., sodium dodecyl sulfate (SDS)), non-ionic detergents (e.g., Triton X-100), and zwitterionic detergents.

[0096]

[0122] Alternatively or additionally, the cellular material of the cornea is removed using enzymatic treatment. Lipase, thermolysin, galactosidase, nuclease, trypsin, endonuclease, and exonuclease can be used to remove cellular material from the cornea. In some embodiments, the cellular material of the cornea is removed using physical techniques. These physical techniques include methods used to dissolve, kill, and remove cells from the tissue matrix through the use of temperature, pressure, and / or electrical disruption. Temperature-based decellularization methods can include rapid freezing protocols. Such temperature-based methods preserve the physical structure of the ECM scaffold. Pressure decellularization involves the controlled use of hydrostatic pressure at high temperatures to avoid unsupervised ice crystal formation that can damage the scaffold. Electrical disruption of the plasma membrane is another option to dissolve the cellular material of the cornea.

[0097]

[0123] In some embodiments, the lenticles can be further processed to exhibit even lower immunoreactivity due to degradation of immunogenic epitopes. This is an important step when using xenogeneic donors. For example, two non-human epitopes that can be present in xenogeneic tissues are N-glycolylneuraminic acid (Neu5GC) and galactose-alpha-1,3-galactose (alpha-Gal). These undesirable epitopes are present not only inside but also on the surface of keratocytes; a minority of epitopes may be buried in glycoaminoglycans (GAGs), also known as mucopolysaccharides, that surround ECM collagen fibrils. In certain embodiments, such epitopes can be removed or their conformation modified (to neutralize immunogens) by enzymatic treatment, such as kinase or galactosidase treatment, and further washing. Alternatively, corneal tissue can be harvested from knockout transgenic pigs that lack the epitopes, thus making it possible to generate non-immunogenic lenticles without the need for a degradation step. In some cases, it may be preferable to also remove the epithelial and / or endothelial cell layer or residue from the lenticule prior to epitope neutralization, which can be done, for example, by scraping with a scalpel or, for example, by rubbing with an abrasive material of suitable coarseness.

[0098]

[0124] In the embodiments described herein, the method of making the decellularized lenticule may further include a sterilization step, which may be combined with packaging and sealing. Sterilization may be performed using a wet agent, gamma radiation, or an electron beam. In one embodiment, sterilization of the decellularized lenticule is performed using an electron beam, as damage to the collagen scaffold is less likely to occur. Alternatively, the radiation utilized to induce crosslink formation may also provide sufficient energy to sterilize the lenticule.

[0099]

[0125] In some embodiments, preparation of the harvested, shaped and decellularized lenticule will include marking the lenticule in a manner that allows the clinician to maintain the proper orientation of the lenticule during re-dissection and laser adjustment of the flap. The marking can be done in a variety of ways, but in all cases will be invisible to the patient once the lenticule is placed. In some embodiments, the marking can be a microscopic incision in the top anterior portion or bottom anterior portion of the lenticule. In some embodiments, the marking can be a line or dot made with dye placed on the top anterior or bottom anterior portion of the lenticule. These methods may also include inscribing one or more asymmetric markers (such as the letter "L") on the periphery of the lenticule to identify the anterior and posterior surfaces of the lenticule.

[0100]

[0126] The invention and embodiments illustrating the methods and materials used may be further understood by reference to the following exemplary protocols. Corneal tissue may be harvested from a porcine donor. The lenticule may be taken from an area within the donor stroma, maintaining dense collagen type I fibrils or Bowman's membrane at the anterior and less dense posterior surfaces of the lenticule. Alternatively, corneal tissue may be taken from the stromal area below the naturally densified surface.

[0101]

[0127] As shown in Figure 1A, a target region of the donor cornea can be dissected into a disk-shaped lenticule 10A having an anterior surface 12, a posterior surface 14, and an organized layer of collagen fibrils 16. At this stage, the lenticule will typically have a diameter of about 0.5 to 10 millimeters and a thickness of less than 250 micrometers.

[0102]

[0128] FIG. 1B shows a lenticle after decellularization, e.g., by chemical, enzymatic, or physical techniques, to create a lenticle in which cellular material is 95-99.99% removed. If desired, the decellularized, shaped lenticle can be further treated to degrade immunogenic epitopes. The lenticle can be further washed and sterilized, and if desired, a crosslinking agent can be applied to the anterior and / or posterior surfaces of the lenticle. The decellularized lenticle (which is typically swollen from application of detergents, surfactants, and / or cleaning solutions) exhibits greater spacing of the fibrillar layers 16. In some embodiments, the upper anterior surface of the lenticle can also be marked with a notch to aid in lenticle orientation in the patient's parenchymal bed.

[0103]

[0129] In Figure 1C, the decellularized (and potentially swollen) lenticule is then subjected to compression to expel excess fluid from the lenticule body and increase collagen density. The collagen fibril layers are compressed together and then at least partially cross-linked to prevent subsequent swelling.

[0104]

[0130] 2, a lenticle 20 is shown having a front region 22 in which the collagen fibril layers are compressed and crosslinked. The bulk region 24 of the lenticle 20 may also be crosslinked to prevent swelling, but not to the same extent as the front region 22.

[0105]

[0131] In Figure 3, a lenticule 30 is shown having one surface (e.g., posterior surface 14) formed with a pattern 32 to promote post-operative integration. The pattern 32 can be formed by selective application of a drug, selective irradiation, or a combination of these techniques. (It will be apparent that the pattern can be applied to the anterior surface 12, the posterior surface 14, or both surfaces.)

[0106]

[0132] FIG. 4A illustrates a lenticule 40A according to the present disclosure in a flat configuration, which is often a typical configuration during manufacturing and / or shipping.

[0107]

[0133] FIG. 4B illustrates the lenticule 40B in an exemplary final curvature state for intracorneal implantation. The lenticule can take a variety of shapes to correct various refractive errors or ocular conditions (discussed further below). Although the lenticule is generally illustrated as a spherical or spheroidal shape, it will be apparent that the lenticule may also be an ellipsoid or any desired shape (e.g., partially toric). Such shapes may be useful for correcting astigmatism or higher order aberrations in the patient's vision. Such aspheric shapes may be useful in matching the shape of the patient's cornea or limbus.

[0108]

[0134] FIG. 5A illustrates a lenticule 50A designed for intracorneal implantation to correct a hyperopic condition. FIG. 5B illustrates a lenticule 50B designed for intracorneal implantation to correct a myopic condition. FIG. 5C illustrates a lenticule 50C designed for intracorneal implantation to correct a presbyopic condition. FIG. 5D illustrates a lenticule 50D designed for intracorneal implantation to correct a condition known as keratoconus, where the natural collagenous structure of the cornea is weakened due to injury, genetics, or other ocular conditions, such as an imbalance of enzymatic or signaling activity in the cornea. The lenticules 50A, 50B, 50C, and 50D are typically designed for intrastromal (between layers of the natural stroma) implantation in the cornea. By placing the lenticule in the stromal tissue 2, an epithelial region 4 and an endothelial region 6 are secured.

[0109]

[0135] FIG. 6A illustrates another embodiment of a lenticule, in which lenticule 60A is fabricated with localized spots 61 of strong cross-linking to create areas of additional mechanical strength, for example, for attachment of surgical sutures during penetrating keratoplasty (PK) or deep lamellar keratoplasty (DALK), as discussed in more detail below.

[0110]

[0136] 6B illustrates yet another embodiment of a lenticule. In this embodiment, lenticule 60B includes a central optically active zone 65 (e.g., having a major diameter dimension of about 3-6.5 millimeters) with moderate crosslinking and a peripheral or peripheral zone 63 with strong crosslinking, again providing an area of ​​additional mechanical strength, e.g., for attachment of surgical sutures or in support of corneal fragile stroma outside the optic zone. Note that zones 63 and 65 need not be concentric, and it may be desirable to offset the optically active zone from the center of the lenticule, e.g., in the treatment of keratoconus.

[0111]

[0137] FIG. 7A illustrates the use of a lenticule 70A according to the present disclosure for a different type of intracorneal transplantation, namely deep lamellar keratoplasty (DALK). In this procedure, an anterior segment of the eye is first removed and a lenticule 70 is placed in the eye to replace a portion of the native Bowman's membrane 3 and stroma 2. The lenticule 70A can be initially secured in place by sutures 7. Optionally, the lenticule 70A can be formed from native donor tissue with an intact Bowman's membrane that is preserved through the decellularization and compaction steps of lenticule formation. Alternatively, the anterior surface of the lenticule can be selectively treated, for example, by shallow radial crosslinking to form a Bowman's membrane-like structure. In either case, following intracorneal transplantation, the patient's peripheral epithelium can grow over the anterior surface of the transplanted lenticule.

[0112]

[0138] 7B illustrates the use of a lenticule 70B that is similar to the DALK lenticule shown in FIG. 7A, but is typically thinner (e.g., less than 200 micrometers) and configured for placement above the patient's intact Bowman's membrane 3 but below the epithelium 4. In this procedure, sometimes referred to as "lamellar keratoplasty," the lenticule 70B can again be secured in place with sutures 7.

[0113]

[0139] FIG 7C illustrates yet another embodiment of a lenticule 70C according to the present disclosure, in this case designed for penetrating keratoplasty (PK) procedures. The lenticule 70C is similar to the DALK lenticule shown in FIG 7A, but is configured to completely replace the entire depth of the central corneal tissue, i.e., the posterior corneal endothelium 6, stroma 2, and Bowman's membrane 3. The lenticule 70C can again be secured in place by sutures 7. Following intracorneal implantation of the lenticule 70C, the patient's peripheral epithelium can grow over the anterior surface of the implanted lenticule.

[0114]

[0140] 8A, 8B, and 8C illustrate two alternative designs for the periphery of a thick lenticule, such as those useful in DALK and PK procedures. In FIG. 8A, a lenticule 80A is shown with a simple, e.g., cylindrical or conical, periphery. In FIG. 8B, a lenticule 80B is shown with a zigzag or stepped edge at the periphery of the lenticule. The shaped edge can be designed to mate with a complementary structure formed in the natural cornea to further aid in bonding of the lenticule 80B to the remaining natural corneal tissue. Stair-step variations (e.g., a reverse zigzag) can also be used. As shown in FIG. 8C, even more sophisticated shapes of the periphery can be used to allow mechanical latch-in or lock-in to the natural cornea. The lock-in feature can reduce or eliminate the need for surgical suturing along the latch-in edge in some cases. The periphery of such a lenticule can be more strongly cross-linked than the central portion.

[0115]

[0141] In some cases, the thickness of these thick lenticles can vary from the center to the edge. For example, the thickness of the lenticles can vary from about 400 micrometers at the center to 550 micrometers at the periphery. This is consistent with the natural thickness variation seen in most corneas, where a typical intact cornea exhibits a central corneal thickness of about 500 micrometers, while the peripheral segments of the cornea can exhibit thicknesses on the order of 550 to about 650 micrometers.

[0116]

[0142] FIG. 9 illustrates diagrammatically a press 90 for use in compressing collagen scaffolds according to the present invention. The press 90 may include a frame 92 and a movable stage 94. The frame holds a top press element 96 and the stage holds a bottom press element 98. At least one of these elements is non-planar and shaped to match the desired final shape of the scaffold. The elements 96 and 98 may be brought into compression by application of a driving force (illustrated diagrammatically by a worm screw 91). In certain embodiments, at least one of the press elements 96, 98 may be transparent so that the scaffold held in compression between the press elements 96 and 98 may be irradiated, for example by a UV radiation source 93, at the same time the scaffold is being molded.

[0117]

[0143] 10A and 10B illustrate a two-part sealed compression and storage container or mold 101 according to the present invention. The container / mold 101 can comprise a mold base 102 and a mold top 104, defining a chamber 105 between the mold base 102 and the mold top 104. The purpose of the mold 101 is to compress a scaffold 100A, e.g., a decellularized stromal collagenous (extracellular matrix) scaffold, and simultaneously remove fluid from the scaffold. FIG. 10A illustrates the mold 101 before compression of the scaffold. A seal 106, e.g., an O-ring or flat gasket seal, initially separates the mold top 104 and the mold bottom 102. In some cases, it may be desirable to fill the remainder of the chamber 105 not occupied by the scaffold 100A with a fluid 107 to avoid gas entrapment in the scaffold during compression. The seal 106 is configured to contain fluid displaced from the scaffolding, as well as any fluid that initially surrounds the scaffolding in the chamber 101. In Figure 5A, the mold is shown before compression, but at its initial moment when the seal 106 creates isolation from the surrounding environment.

[0118]

[0144] The top portion 104 of the mold can be at least partially transparent or semi-transparent to allow actinic radiation to pass through the scaffold to crosslink it. Alternatively, or in addition, the bottom portion 102 of the mold can be transparent to actinic radiation. The transparent portion of the mold can be made of plastic, glass, ceramic, or metal, or any combination of such materials, so long as the mold is sufficiently transparent or semi-transparent to allow radiation transmission.

[0119]

[0145] FIG. 10B illustrates the mold 101 after compression of the scaffold 100B. A seal, e.g., O-ring 106, can move within groove 103, which then contains the expelled fluid. (Various other drainage mechanisms can also be used.) In this state, the mold 101 can be used for crosslinking and sterilization. If the appropriate wavelength and fluence of radiation is selected, such sterilization can be performed simultaneously with crosslinking. (In many cases, the radiation dose required to inactivate the virus will be much less than that required for crosslinking.) The compressed and sterilized mold can be further used for shipping or long-term storage. In the case of active crosslinking radiation, e.g., UV radiation, the radiation needs to reach all surfaces of the lenticule during this process to be effective for sterilization purposes as well. It may therefore be desirable to use a seal, e.g., an O-ring or flat gasket that is transparent to the selected active radiation. For example, if UV light is used to both crosslink and sterilize the lenticule during molding, the seal can be made from a UV transparent or semi-transparent material, such as a fluoropolymer, e.g., polytetrafluoroethylene (PTFE), fluorinated ethylene propylene (FEP), ethylene tetrafluoroethylene (ETFE), or perfluoroalkoxy (PFA) composition.

[0120]

[0146] Matching the shape of the scaffold or lenticule before compression to the shape of the mold typically ensures that the density of the lenticule remains uniform after compression. For example, compression can halve the vertical dimension and uniformly double the density.

[0121]

[0147] 11 illustrates a further device 110 according to the invention for stretching a scaffold 161, which may be a natural decellularized parenchymal harvest or a scaffold that has been decellularized and subjected to an initial compression process. In this embodiment, the scaffold 161 is attached to the opening of a chamber 163 by a sealing mechanism 167, for example by crimping the periphery of the scaffold 161 between two flat flanges. (Various other sealing mechanisms can be used instead of or in addition to crimping to form a fluid-tight connection between the scaffold and the opening of the chamber.) A fluid 166 is then introduced into the chamber 163 via a controlled pressure conduit 165 to fill the interior of the chamber and apply pressure to the scaffold 161. For example, horizontal and / or tangential extension can be obtained by maintaining sufficient hydrostatic pressure in the chamber, for example, from about 30 to about 500 mbar, preferably from about 50 to about 200 mbar (or from about 22.5 to about 375 Torr, preferably from about 37.5 to about 150 Torr).

[0122]

[0148] Even if the fluid applying the mechanical stress to the scaffold is pure water or balanced salt solution (BSS), the application of this fluid pressure can also cause the scaffold to deswell (release free water). Fluid can flow out of the scaffold in any direction (e.g., into a pressurized fluid chamber or by seepage from the outer surface of the scaffold, or both). Hypertonic or hypotonic fluids can also be used in chamber 163 to enhance or limit this effect.

[0123]

[0149] Exposing the ECM scaffold to this type of stretching can cause the collagen fibrils to align and construct a curved shape. When the desired curvature is achieved, the shape can be secured (or fixed) by crosslinking, e.g., by actinic radiation 168, as shown.

[0124]

[0150] In Fig. 12 yet another alternative device 120 is shown similar to that of Fig. 6 but with the addition of a compression applying plate element 171 for simultaneous stretching and compression of the scaffold and to facilitate exposure to crosslinking radiation. Horizontal and / or tangential stretching is again provided by maintaining sufficient hydrostatic pressure in the chamber of about 30 to about 500 mbar, preferably about 50 to about 200 mbar. Vertical / radial compression can be applied simultaneously by the top plate 171. Preferably, the top plate 171 is at least partially transparent, e.g. clear or semi-transparent, to allow the actinic radiation to pass through the top plate to the scaffold to crosslink it.

[0125]

[0151] Although the top plate 171 is illustrated as a flat plate, the topography of this compressible element can take on any desired shape and / or curvature to ensure that the collagen fibril configuration is properly fixed to build a lenticule of the desired shape, which may again be ensured or fixed by radiation-induced crosslinking.

[0126]

[0152] FIG. 13 illustrates an apparatus 130 for measuring the transparency of a lenticle made according to the present disclosure. The apparatus 130 includes a light source 132, a cuvette 134 (in which the lenticle 135 can be placed), a lens 136, and a detector 138. The light source, e.g., a light emitting diode (LED) with a waveguide / fiber and a beam collimator (not shown), generates a beam of preferably collimated light rays 133. The beam should be spatially coherent with a flat (non-Gaussian) intensity profile over its beam width D (e.g., about 5-7 mm). For example, the light source 132 can generate green light having a central wavelength of about 500 nanometers and a bandwidth of about 5-10 nanometers.

[0127]

[0153] Light from the light source 132 is directed into a cuvette 134 in which a lenticle 135 is suspended in an index-matching fluid 137 (e.g., a silicone oil or other fluid that is immiscible with water and chemically inert, with a refractive index n of approximately 1.376). The value of n=1.376 represents the nominal or average refractive index of natural corneal tissue. In some cases, it may be advantageous to have a lenticle of a different refractive index. In such cases, the fluid in the cuvette will be selected to match the particular n value of the lenticle under test. The light passing through the cuvette 134 (and lenticle 135) is then directed into lens 136, which is a high-quality lens with no spherical aberration at the selected beam wavelength. Lens 136 has a focal length f and serves to direct the focused light to a detector 138 located at the focal plane of lens 136. If the transparency of the lenticle were perfect (assuming ideal optical transparency for the optical elements through which light from the source 132 passes to the detector 138), the size of the beam impinging between the detectors (located at the focal plane) would be a diffraction-limited spot of uniform intensity. In reality, the lenticle is never perfectly transparent, and the beam intensity image at the detector will exhibit some haze due to forward scattering of light within the scaffold. (The focal length of the lens, together with the beam diameter, must be selected to impose the desired diffraction-limited angular resolution.)

[0128]

[0154] The transparency of the lenticule can therefore be assessed by measuring the intensity profile of the detected beam. Suitable photodetectors for measuring the intensity or brightness profile can include, for example, a photographic plate, a CCD array or a scanning pinhole detector.

[0129]

[0155] FIG. 14 illustrates such a method of quantifying transparency by measuring the scattering angle (referred to herein as "Θ" or "theta") of the lenticle. FIG. 14 illustrates an intensity distribution curve measured at the focal plane of the lens. This Gaussian distribution represents the sum of all the contributions of the scattering wavefront. The more scattering at the lenticle, the wider the detected beam. One measure of scattering (reduction in optical transparency) is the full width at vertical half maximum (FWHV), as shown in FIG. 14. It should be understood that the Gaussian curve in FIG. 14 is idealized, and that the actual intensity curve may be distorted by noise or other spurious signals. Multiple measurements and averaging (or other known noise-reducing signal processing techniques) can be used to obtain the best data representation of scattering by the lenticle. The FWHV value divided by the focal length f provides a measure of the angular divergence (in radians) of the beam. Θ=FWHV / f[rad]

[0130]

[0156] Theta depends only on the amount of scattering and can also be expressed in arc minutes using the conversion: 1 arcmin = 291 microrad. Lenticles produced by the methods disclosed herein exhibit satisfactory transparency when the angle Θ is less than 4 arc minutes, preferably less than 3 or 2 arc minutes, and more preferably in some cases less than 1 arc minute (e.g., for intracorneal implants).

[0131]

[0157] All patents or publications cited herein are incorporated by reference in their entirety. Although the applicant's teachings are described in conjunction with various embodiments, it is not intended that the applicant's teachings be limited to such embodiments. On the contrary, the applicant's teachings encompass various alternatives, modifications, and equivalents, as will be understood by those skilled in the art. Any element or feature shown in connection with one embodiment can be used in exchangeable combination with or in addition to any element or feature shown in any other embodiment, and all such permutations of elements and features should be understood to be encompassed by the present disclosure. Therefore, it should be understood that the present invention is not to be limited to the embodiments disclosed herein, but should be understood from the following claims, which should be interpreted as broadly as permitted under law.

Claims

1. 1. A method for forming a scaffold from donor corneal stroma extracted from a central region of a donor corneal source, comprising: decellularizing the donor corneal stroma to obtain a scaffold; Removing fluid present in the scaffold; and Cross-linking at least a portion of the scaffold to prevent subsequent swelling. The method includes:

2. performing lenticule extraction on the cornea using a keratome, laser, or water jet to obtain donor corneal stroma; The method of claim 1 further comprising:

3. 10. The method of claim 1, further comprising shaping the scaffold, wherein the shaping step is performed simultaneously or sequentially with the extraction of the donor corneal stroma.

4. The method of claim 1 , wherein the step of removing fluid further comprises applying pressure, acceleration (e.g., centrifugal force) or vacuum to the scaffold.

5. 2. The method of claim 1, wherein the decellularizing step further comprises treating the scaffold with a chemical decellularizing agent, and optionally the decellularizing step is performed before or after a water removing step or before or after a crosslinking step.

6. 6. The method of claim 5, wherein the decellularizing step further comprises removing cellular debris from the scaffold using a detergent or surfactant.

7. 10. The method of claim 1, further comprising enzymatically removing or modifying the conformation of at least one immunogenic epitope of the scaffold.

8. 10. The method of claim 1, wherein the steps of removing fluid from the scaffold and crosslinking the scaffold result in at least a portion of the scaffold having a greater collagen density than the original harvested stroma.

9. 10. The method of claim 1, wherein the step of removing fluid from the scaffold further comprises compressing the scaffold, whereby at least a portion of the compressed and crosslinked scaffold has a composition of at least 15 percent collagen, or at least 30 percent collagen, or at least 25 percent collagen, or at least 60 percent collagen.

10. 10. The method of claim 1, wherein the step of crosslinking further comprises exposing at least a portion of the compressed scaffold to a crosslinking promoter or actinic radiation.

11. the cross-linking step further comprises exposing at least a portion of the compacted scaffold to radiation to induce cross-linking via peptide bond formation between collagen fibrils, with or without the assistance of a cross-linking promoter or other energy mediator; Optionally, the step of crosslinking the scaffold further comprises irradiating the scaffold with ultraviolet radiation, x-rays, gamma radiation, or an electron beam. The method of claim 10.

12. 11. The method of claim 10, wherein the crosslinking step further comprises exposing at least a portion of the compressed scaffold to ultraviolet radiation by direct exposure, or by exposure at a small incidence angle, or via an evanescent waveguide connected to a surface of the scaffold.

13. 11. The method of claim 10, wherein the step of crosslinking further comprises selectively exposing surface portions of the compressed scaffold to radiation such that the surface portions exhibit a greater degree of crosslinking and higher collagen density than bulk regions of the scaffold.

14. 11. The method of claim 10, wherein the step of crosslinking further comprises applying radiation sufficient to inactivate any microbial agents and / or sterilize the scaffold.

15. The method of claim 1 , wherein the scaffold is configured for use as an implantable lenticule having a lenticule body, an anterior surface, and a posterior surface that give the lenticule a final desired shape.

16. 2. The method of claim 1, wherein the step of crosslinking further comprises treating at least a portion of the posterior surface of the lenticule with a crosslinking agent or by selective application of patterning radiation to promote adhesion of the lenticule to a stromal bed of a patient when implanted therein.

17. The method of claim 1 , wherein the crosslinking step further comprises enhancing the optical transparency of the scaffold.

18. 18. The method of claim 17, wherein the scaffold exhibits a scattering angle theta of less than 4 arcmin.

19. 18. The method of claim 17, wherein the lenticule exhibits a scattering angle theta of less than 3 arc minutes or less than 2 arc minutes.

20. 20. The method of claim 17, wherein the lenticule exhibits a scattering angle theta of less than 1 arc minute.

21. 1. A method of reducing radiation application to form a crosslinked collagenous scaffold, comprising: Removing fluid present in the scaffold; and subjecting the compacted scaffold to radiation to crosslink at least a portion of said scaffold, thereby defining a final desired shape and preventing subsequent swelling when said scaffold is exposed to an aqueous environment. The method includes:

22. 22. The method of claim 21, further comprising compressing the scaffold by applying pressure to at least one surface of the scaffold.

23. The step of compressing the scaffold further comprises: and further comprising: securing the scaffold across an opening of a fluid chamber and applying a fluid pressure to one surface of the scaffold to form a scaffold of a first desired shape.

23. The method of claim 22.

24. 23. The method of claim 22, further comprising the step of decellularizing the scaffold prior to compaction or vice versa.

25. 22. The method of claim 21, wherein the step of crosslinking the scaffold further comprises irradiating the scaffold with ultraviolet radiation, x-rays, gamma radiation, or an electron beam.

26. The step of irradiating the scaffold comprises irradiating the scaffold with about 15 Joules / cm ２ ~ about 2500 Joules / cm ２ and optionally about 15 Joules / cm ２ ~ 600 Joules / cm ２ 26. The method of claim 25, further comprising irradiating with UV-C radiation (from about 100 nm to about 280 nm) having a fluence ranging from about 240 nm to about 280 nm, and preferably in some cases with UV-C radiation from about 240 nm to about 280 nm, to induce crosslinking.

27. 22. The method of claim 21, wherein the crosslinking step further comprises enhancing the optical transparency of the scaffold.

28. 28. The method of claim 27, wherein the scaffold exhibits a scattering angle, theta, of less than 4 arc minutes, or less than 3 arc minutes, or less than 2 arc minutes, or less than 1 arc minute.

29. 22. The method of claim 21, further comprising compressing the scaffold such that the scaffold exhibits a collagen concentration of greater than 15%, or greater than 30%, or greater than 45%, or greater than 60%.

30. 22. The method of claim 21, wherein the scaffold exhibits a residual amount of cellular material, as assessed by residual DNA or RNA content, that is less than 1 weight percent, or less than 0.1 weight percent, or less than 0.01 weight percent of the original DNA or RNA content.

31. a lenticule body derived from a corneal donor source having an anterior surface and a posterior surface that provide the lenticule with a desired shape; the lenticule body further comprising collagen that is at least partially cross-linked to prevent axial swelling. Collagenous lenticules, including:

32. 32. The collagenous lenticule of claim 31, wherein the collagen layer is crosslinked by application of radiation, said collagenous lenticule being further characterized by induced chemical bonds between collagen fibrils.

33. The collagenous lenticule of claim 31, which has been decellularized such that the amount of cellular material remaining in the collagenous lenticule, as assessed by residual DNA or RNA content, is less than 1 weight percent, or less than 0.1 weight percent, or less than 0.01 weight percent of the original DNA or RNA content.

34. 32. The collagenous lenticule of claim 31 , formed in a desired shape so that it can be implanted into the stromal region of a patient's eye to alter the refractive power of the cornea.

35. 32. The collagenous lenticule of claim 31, having a disk-like shape and a diameter of about 0.5 mm to about 10 mm, optionally with a curvature obtained by applying pressure to at least one side of said collagenous lenticule.

36. 32. The collagenous lenticule of claim 31 having a maximum thickness of less than about 600 micrometers, less than about 400 micrometers, or less than about 200 micrometers, or less than about 100 micrometers, or less than 50 micrometers.

37. 32. The collagenous lenticule of claim 31, which exhibits low immune reactivity due to removal or degradation of immunogenic epitopes.

38. The collagenous lenticule of claim 31 , wherein the posterior surface of the collagenous lenticule further comprises a crosslinking agent or a pattern of variable crosslink formation to promote attachment of the collagenous lenticule to a stromal bed when implanted into a patient's stromal bed.

39. 32. The collagenous lenticule of claim 31, wherein the anterior surface further comprises a anterior surface region having a greater collagen density than a bulk region of the lenticule body.

40. The collagenous lenticule of claim 31 , having a curvature optionally formed by applying pressure to one surface of the scaffold.

41. 32. The collagenous lenticule of claim 31 having sufficient optical clarity for use as an intracorneal implant.

42. 42. The collagenous lenticule of claim 41, exhibiting a scattering angle, theta, of less than 3 arc minutes, or less than 2 arc minutes, or less than 1 arc minute.

43. 32. The collagenous lenticule of claim 31, which has been reduced in fluid and optionally compressed to exhibit a collagen concentration of greater than 15%, or greater than 30%, or greater than 45%, or greater than 60%.

44. 42. The collagenous lenticule of claim 41, which has been sterilized by radiation, such as UV radiation or electron beam.

45. 32. The collagenous lenticule of claim 31, further comprising a container surrounding the collagenous lenticule for storage and / or shipping.

46. 46. ​​The collagenous lenticule of claim 45, wherein the container also functions as a mold for shaping, compressing the scaffold or for removing fluids from the scaffold.

47. 46. ​​The collagenous lenticule of claim 45, wherein the container further comprises at least one radiolucent or semi-permeable surface to facilitate crosslinking or sterilization of the scaffold.

48. 32. The collagenous lenticule of claim 31, exhibiting a refractive index greater than that of natural corneal stromal tissue.

49. 32. The collagenous lenticule of claim 31, exhibiting a refractive index greater than 1.

377.

50. 32. The collagenous lenticule of claim 31, exhibiting a refractive index greater than 1.

378.

51. 32. The collagenous lenticule of claim 31 exhibiting a refractive index gradient.

52. a lenticule body derived from a corneal donor source having an anterior surface and a posterior surface that provide a desired shape for the lenticule; Sufficient optical clarity for use as an intracorneal implant A decellularized collagenous lenticule comprising:

53. 53. The decellularized collagenous lenticule of claim 52, exhibiting a scattering angle, theta, of less than 4 arcminutes.

54. 53. The decellularized collagenous lenticule of claim 52, exhibiting a scattering angle, theta, of less than 2 arcmin.

55. 53. The decellularized collagenous lenticule of claim 52, exhibiting a scattering angle, theta, of less than 1 arc minute.

56. The lenticular body is At least partially cross-linked collagen that is at least partially defluidized and optionally compressed to prevent axial swelling 53. The decellularized collagenous lenticule of claim 52, further comprising:

57. 1. A storage container for a lenticule comprising an upper body and a lower body defining a sealed cavity, at least a portion of the upper body or at least a portion of the lower body, or at least a portion of both bodies, is radiolucent to allow for in situ sterilization of the lenticule; Storage container.

58. 58. The lenticule storage container of claim 57, wherein at least one seal is provided between said upper body and said lower body, optionally said seal being an O-ring or flat gasket seal.

59. 60. The lenticule storage container of claim 58, wherein the seal is radiolucent.

60. 60. The lenticule storage container of claim 58, wherein the seal is UV transparent and optionally comprises a fluoropolymer composition.

61. 59. The lenticule storage container of claim 58, wherein the seal comprises a composition of polytetrafluoroethylene (PTFE), fluorinated ethylene propylene (FEP), ethylene tetrafluoroethylene (ETFE), or perfluoroalkoxy (PEA).

Images